Endocrine Physiology

1. Endocrine Physiology Dale Buchanan Hales, PhD Department of Physiology & Biophysics

2. Claude Bernard: the father of endocrinology More than 100 years ago Claude Bernard stated that the endocrine system regulates the internal milieu of an animal. The “internal secretions” or hormones were liberated by one part of the body, traveled via the bloodstream to distant targets cells.

3. Endocrine system maintains homeostasis The concept that hormones acting on distant target cells to maintain the stability of the internal milieu was a major advance in physiological understanding. The secretion of the hormone was evoked by a change in the milieu and the resulting action on the target cell restored the milieu to normal.The desired return to the status quo results in the maintenance of homeostasis

4. Sensing and signaling Endocrine “glands” synthesize and store hormones. These glands have a sensing and signaling system which regulate the duration and magnitude of hormone release via feedback from the target cell.

5. Endocrine vs. Nervous System • Major communication systems in the body • Integrate stimuli and responses to changes in external and internal environment • Both are crucial to coordinated functions of highly differentiated cells, tissues and organs • Unlike the nervous system, the endocrine system is anatomically discontinuous.

6. Principal functions of the endocrine system • Maintenance of the internal environment in the body (maintaining the optimum biochemical environment). • Integration and regulation of growth and development. • Control, maintenance and instigation of sexual reproduction, including gametogenesis, coitus, fertilization, fetal growth and development and nourishment of the newborn.

7. Hormones travel via the bloodstream to target cells

8. Types of cell-to-cell signaling Classic endocrine hormones travel via bloodstream to target cells; neurohormones are released via synapses and travel via the bloostream; paracrine hormones act on adjacent cells and autocrine hormones are released and act on the cell that secreted them. Also, intracrine hormones act within the cell that produces them.

9. A cell is a target because is has a specific receptor for the hormone

10. Major hormones and systems • Top down organization of endocrine system. • Hypothalamus produces releasing factors that stimulate production of anterior pituitary hormone which act on peripheral endocrine gland to stimulate release of third hormone • Specific examples to follow • Posterior pituitary hormones are synthesized in neuronal cell bodies in the hypothalamus and are released via synapses in posterior pituitary. • Oxytocin and antidiuretic hormone (ADH)

11. Types of hormones-1 • Amine hormones: • Hormones derived from the amino acid tyrosine. • These include epinephrine, norepinephrine and thyroid hormone. • Epinephrine and norepinephrine are produced by the adrenal medulla –water soluble • Thyroid hormone is produced by the thyroid gland –lipid soluble

12. Types of hormones -2 • Peptide/protein hormones: • Range from 3 amino acids to hundreds of amino acids in size. • Often produced as larger molecular weight precursors that are proteolytically cleaved to the active form of the hormone. • Peptide/protein hormones are water soluble. • Comprise the largest number of hormones– perhaps in thousands

13. Types of hormones-3 • Steroid hormones: • All steroid hormones are derived from cholesterol and differ only in the ring structure and side chains attached to it. • All steroid hormones are lipid soluble • 1,25-dihydroxy Vitamin D3 is also derived from cholesterol and is lipid soluble

14. Peptide/protein hormones • Are encoded by a specific gene which is transcribed into mRNA and translated into a protein precursor called a preprohormone • Preprohormones are often post-translationally modified in the ER to contain carbohydrates (glycosylation) • Preprohormones contain signal peptides (hydrophobic amino acids) which targets them to the golgi where signal sequence is removed to form prohormone • Prohormone is processed into active hormone and packaged into secretory vessicles

15. Peptide/protein hormones • Secretory vessicles move to plasma membrane where they await a signal. Then they are exocytosed and secreted into blood stream • In some cases the prohormone is secreted and coverted in the extracellular fluid into the active hormone: an example is angiotensin is secreted by liver and converted into active form by enzymes secreted by kidney and lung

16. Peptide/protein hormone synthesis

17. Steroid hormones • Are not packaged, but synthesized and immediately released • Are all derived from the same parent compound: Cholesterol • Enzymes which produce steroid hormones from cholesterol are located in mitochondria and smooth ER • Steroids are lipid soluble and thus are freely permeable to membranes so are not stored in cells

18. Steroid hormones • Steroid hormones are not water soluble so have to be carried in the blood complexed to specific binding globulins. • Corticosteroid binding globulin carries cortisol • sex steroid binding globulin carries testosterone and estradiol • In some cases a steroid is secreted by one cell and is converted to the active steroid by the target cell: an example is androgen which secreted by the gonad and converted into estrogen in the brain

19. Steroid hormone synthesis All steroid hormones are derived from cholesterol. A series of enzymatic steps in the mitochondria and ER of steroidogenic tissues convert cholesterol into all of the other steroid hormones and intermediates. 1,25-dihydroxy Vitamin D3 is also derived from cholesterol

20. Steroids can be transformed to active steroid in target cell

21. Amine hormones: derived from tyrosine • Epinephrine, norepinephrine and dopamine are water soluble and are synthesized and secreted like peptide hormones • Thyroid hormones are produced by modification of a tyrosine residue contained in thyroglobulin, post-translationally modified to bind iodine, then proteolytically cleaved and released as T4 and T3. T3 and T4 then bind to thyroxin binding globulin for transport in the blood

22. Synthesis of catecholamines

23. Thyroid hormones

24. Regulation of hormone secretion • Sensing and signaling: a biological need is sensed, the endocrine system sends out a signal to a target cell whose action addresses the biological need. Key features of this stimulus response system are: ·        receipt of stimulus ·        synthesis and secretion of hormone ·        delivery of hormone to target cell ·        evoking target cell response ·        degradation of hormone

25. Feedback control • Negative feedback is most common: for example, LH from pituitary stimulates the testis to produce testosterone which in turn feeds back and inhibits LH secretion • Positive feedback is less common: examples include LH stimulation of estrogen which stimulates LH surge at ovulation

26. Substrate-hormone control • glucose and insulin: as glucose increases it stimulates the pancreas to secrete insulin

27. Neural control • Neural input to hypothalamus stimulates synthesis and secretion of releasing factors which stimulate pituitary hormone production and release

28. Chronotropic control • Endogenous neuronal rhythmicity • Diurnal rhythms, circadian rhythms (growth hormone and cortisol), Sleep-wake cycle; seasonal rhythm

29. Inputs to endocrine cells

30. Circadian (chronotropic) control

31. Circadian Clock

32. Negative feedback effects of cortisol

33. Feedback control of insulin by glucose concentrations

34. Hormone-Receptor interactions • Definition: a protein that binds a ligand with high affinity and low capacity. This binding must be saturuable. • A tissue becomes a target for a hormone by expressing a specific receptor for it. Hormones circulate in the blood stream but only cells with receptors for it are targets for its action.

35. Hormone binding study

36. Hormone-receptor interactions • Hormone--receptor interaction is defined by an equilibrium constant called the Kd, or dissociation constant. • The interaction is reversible and how easily the hormone is displaced from the receptor is a quantitation of its affinity. • Hormone receptor interactions are very specific and the Kd ranges from 10-9 to 10-12 Molar

37. Analysis of receptor interactions • Free hormone, R=unbound receptor, HR=hormone receptor complex; Kd = dissociation constant • Kd is the concentration of hormone occupying 50% of the receptors are equilibrium • This relationship predicts that as the number of receptors increases then hormone concentration can decrease to maintain the HR complex at the same level.

38. Analysis of hormone interactions

39. Analysis of hormone interactions: Scatchard plots

40. Spare receptors • The "spare receptor" concept is based on this relationship and is states that maximum biological activity is often obtained when only 5 to 10% of the receptors are bound. • When the number of spare receptors are decreased maximum biological activity is achieved at a higher concentration of hormone. Therefore, the greater the number of receptors, the greater the sensitivity of them to hormone

41. Binding vs. biological response Spare receptors Amplification by 2nd messenger

42. Hormonal measurements • Bioassay • an assay system (animal, organ, tissue, cell or enzyme system) is standardized with know amounts of the hormone, a standard curve constructed, and the activity of the unknown determined by comparison • example: testosterone stimulates growth of prostate gland of immature or castrate rat in a dose-dependent manner. Androgen content of unknown sample can be determined by comparison with testosterone. • disadvantage: cumbersome and difficult • advantage: measures substance with biological activity, not just amount

43. Original bioassay systems defined the endocrine system • Remove endocrine gland and observe what happened • Prepare crude extract from gland, inject back into animal and observe what happened • In isolated organ or cell systems, add extract or purified hormonal preparations and measure biological response

44. Hormonal measurements • Chemical methods • chromatography • spectrophotometery

45. Radioimmunoassay • Radioactive ligand and unlabeled ligand compete for same antibody. Competition is basis for quantitation • saturate binding sites with radioactively labeled hormone (ligand) • incubate complex with increasing amounts of cold (unlabeled) ligand to establish standard curve • in parallel incubate complex with unknown and determine its concentration by comparison • cold ligand (standard or unknown) competes with labeled ligand for binding to antibody and displaces it in a dose-dependent way • immunoprecipitate ligand-antibody complex and determine radioactivity • amount of cold ligand is inversely proportional to amount of radioactivity • (cold competes with hot so the more cold that binds antibody the more hot is displaced resulting in fewer counts being associated with complex.

46. RIA • advantages: • extremely sensitive due to use of radioisotope • large numbers of samples can be processed simultaneously • small changes in hormone concentrations can be reproducibly quantitated • Easily automated for high-throughput analysis • disadvantage: • can't determine if hormone measured has biological activity • peptide hormones can be denatured and not active but still retain their antigenic character

47. RIA radioactivity Increasing amount of insulin

48. Classes of hormones • The hormones fall into two general classes based on their solubility in water. • The water soluble hormones are the catecholamines (epinephrine and norepinephrine) and peptide/protein hormones. • The lipid soluble hormones include thyroid hormone, steroid hormones and Vitamin D3

49. Water soluble hormones • Water soluble hormones are synthesized in endocrine cells and packaged into secretory granules. • These granules fuse with the plasma membrane in an energy dependent process called exocytosis and are released into the extracellular fluid surrounding the cell. • Water soluble hormones are freely soluble in the bloodstream and can travel to their targets without being bound to blood proteins– however, many are associated with binding proteins that modulate their bioavailability

50. Lipid soluble hormones • Lipid soluble hormones are synthesized in endocrine cells but are not packaged in secretory granules. • Lipid soluble hormones passively diffuse out of the endocrine cell and become complexed with blood proteins called globulins. • The hormone protein complex is carried by the blood stream to the target cells where the hormone is released from the protein which enables it to bind to the target cell receptor. • Some lipid soluble proteins don’t have binding proteins, for example, aldosterone.